Journal: Oncotarget

Article Title: FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells

doi: 10.18632/oncotarget.7672

Figure Lengend Snippet: Protein-DNA complexes from MDA-MB-231 cells were cross-linked by using formaldehyde and sonicated. ( A , B ) Chromatin fragments from these cells were immunoprecipitated (with antibodies specific to RNA polymerase II (immunoprecipitation 1 [IP-1]; positive control), mouse IgG (IP-2; negative control), and FOXM1 (IP-3) as indicated. Input, total DNA. After reversal of cross-linking, the immunoprecipitated DNA was amplified by PCR using the specific primers and resolved on 1.2% agarose gels. ChIP assay demonstrated that FOXM1 protein bound to the promoter region of the eEF2K gene. ( C ) To confirm this finding, immunoprecipitated DNA was analyzed by PCR with primers specific to eEF2K gene promoter fragment containing BS7. The assay results confirmed the binding of FOXM1 to the eEF2K gene promoter. ( D , E ) Cells were transfected with 50 nM FOXM1 siRNA or control siRNA (D) or with a FOXM1 expression vector and pRTLK vector (renilla) (E) and either the pGL3 empty vector or the pGL3-EF2K vector. Cells were harvested 40 h after transfection, and protein extracts were preapared and analyzed for dual-luciferase activity. Triplicate plates were used to calculate the mean fold induction of transcriptional activity. The luciferase activity values are relative to the activity of the cotransfected renilla luciferase. The luciferase reporter assay demonstrated that inhibition of FOXM1 expression led to down-regulation of eEF2K activity and that expression of FOXM1 resulted in increased eEF2K activity. The data are means with standard deviations. *represents significant difference between indicated groups.

Article Snippet: Following treatment, total cellular RNA was isolated from the collected cells with TRIzol Reagent (Life Technologies, Carlsbad, CA), and complementary DNA (cDNA) was obtained from 1 μg of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Life Technologies) The cDNAs for FOXM1B, FOXM1C, eEF2K and GAPDH were amplified using the Platinum Taq DNA Polymerase kit (Life Technologies) with specific gene primers.

Techniques: Sonication, Immunoprecipitation, Positive Control, Negative Control, Amplification, Binding Assay, Transfection, Control, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Inhibition